Azheimer's disease is a degenerative disorder of the central nervous system which causes memory impairment and cognitive loss during mid to late life. The disease is characterized by two primary pathological features, extracellular amyloid plaques in the brain, and intra-neuronal neurofibrillary tangles. These lesions inhibit neuronal and glial cell function, and lead to synaptic loss and dementia. Both early and late onset forms of the disease have been shown to have genetic components, and four genes have been definitively associated with increased risk for AD: APP, PS1, PS2 and ApoE. These genes are functionally linked by their roles in the production, transport, and/or elimination of, amyloid-β(Aβ), the primary constituent of Alzheimer's amyloid plaques (reviewed in Selkoe, D. 1999, Nature 399 supp: A23).
Alzheimer's amyloid plaques are comprised largely of the 40–42 amino acid peptide Aβ (Glenner, G. G., and Wong, C. W., 1984 Biochem. Biophys. Res. Commun. 122:1131). Aβ is derived by proteolytic cleavage from the b-Amyloid Precursor Protein, or βAPP (Kang J. et al. 1987, Nature 325:733). Three secretase activities cleave APP to generate the Aβ peptide or a shorter, alternative cleavage product called p3. β-secretase generates the N-terminus of Aβ, while α-secretase cleaves internal to Aβ sequences to generate the N-terminus of p3. γ-secretase cleaves the C-terminal β and α secretase products of APP to generate the heterogeneous C-terminal ends of Aβ and p3. APP mutations found in familial Alzheimer's disease (FAD) pedigrees are clustered around the three secretase cleavage sites (Goate, A., et al. 1991, Nature 349:704; Murrell, J., et al. 1991, Science 254: 97; Chartier-Harlin et al. 1991, Nature 353: 844; Mullan, M. et al. 1992, Nature Genet. 1: 345; Levy, E. et al., 1990, Science 248: 1124; Hendriks, L. et al. 1992, Nature Genet. 1:218) and they each increases total Aβ (Aβ42+Aβ40) or increases the Aβ 42/40 ratio. Since Aβ42 precipitates more readily in vitro and is the primary component of early forms of amyloid deposits called diffuse plaques, it has been postulated that increased systemic Aβ42 could lead to earlier formation of plaque, and earlier onset of AD.
Family studies identified two other genes, presenilin-1 (PS1) and presenilin-2 (PS2), associated with dominantly inherited, early onset AD, (Sherrington, R. et al. 1995, Nature 375: 754; Levy-Lahad, E. et al. 1995, Science 269: 973; Rogaev., E. I. et al. 1995, Nature 376: 775). These proteins are similar to each other in sequence and encode polytopic membrane proteins with 8 transmembrane segments. Studies in FAD human cell lines, in transfected cells, and in transgenic mice have demonstrated that the PS FAD mutations cause a change in the processing pattern of APP, resulting in an increased ratio of Aβ 42/40 (Scheuner, D. et al. 1996, Nat. Med. 2: 864; Citron, M. et al. 1997, Nat. Med. 3:67; Borchelt, D. et al. 1996, Neuron 17: 1005; Duff, K. et al. 1996, Nature 383: 710; Tomita, T. et al. 1997, PNAS 94:2025). Studies on PS1 knockout mice demonstrated that loss of PS1 function leads to reduction in Aβ production due to a reduction of γ-secretase activity (De Strooper, B. et al. 1998, Nature 391: 387). Presenilin function is thus implicated in the activity of γ-secretase in two ways: missense mutations alter g-secretase cleavage specificity, while loss of presenilin activity leads to loss of γ-secretase activity.
Inhibition of presenilin activity decreases Aβ production and is thus a potentially useful therapeutic approach to Alzheimer's disease. However, despite the functional link to γ-secretase activity and the generation of Aβ, the biochemical nature of PS activity is poorly understood. Various functions have been proposed, including action in the ER and/or Golgi complex as a chaperone for APP, Notch, and/or γ-secretase (Thinakaran, G. et al. 1998, Neurobiol. Dis. 4: 438), activity as a novel aspartyl protease, i.e. as g-secretase itself (Wolfe, M. S. et al. 1999, Nature 398: 513), and potential roles in the response to oxidative stress and apoptosis (Wolozin, B. et al. 1996, Science 274:1710; vito, P. et al. 1997, J. Biol. Chem 272: 28315; Guo, Q., et al. 1997, J. Neurosci. 17: 4212). The absence of a clear functional assay increases the difficulty of designing useful small molecule therapeutics targeted at presenilin. An alternative strategy to targeting presenilin is to discover additional proteins which act together with presenilins in the pathway of γ-secretase and Aβ production and which might be more amenable to drug development. One useful method for the discovery of such novel targets is to perform genetic screens in model organisms such as Drosophila and C. elegans for genes that interact with presenilins.
Invertebrate orthologues of the PS genes have been identified by both sequence searches and genetic screens. The C. elegans genome contains three presenilin genes, sel-12 (suppressor and/or enhancer of lin-12; Levitan, D. et al. 1995, Nature 377:351), hop-1 (homolog of presenilin; Li, X. et al, 1997, PNAS 94:12204) and spe-4 (spermatogenesis defective; L'Hernault et al., 1992, J. Cell Biol. 119:55). sel-12, hop-1 and spe-4 have 48, 35 and 23% sequence similarity, respectively, to PS1 and 2. sel-12 and hop-1 have overlapping functions in several tissues (see below), while spe-4 appears to perform an independent function in the male germ line. Rescue experiments using transgenes have shown that human PS1 and PS2 can rescue phenotypes caused by loss of sel-12, demonstrating that presenilin function has been conserved from nematodes to mammals (Levitan, D. et al. 1996, Nature 377:351; Baumeister, R. et al. 1997, Genes Function 1: 149).
Sel-12 was identified genetically as a suppressor of an activated allele of the Notch gene lin-12. This discovery established a functional link between presenilin activity and activity of the Notch signaling pathway. In vivo experiments in mice (Herreman, A. et al. 1999, PNAS 96:11872), Drosophila (Struhl, G. et al. 1999, Nature 398: 522; Ye, Y. et al. 1999 Nature 398:525) and C. elegans (Li, X. et al, 1997, PNAS 94:12204; Westlund, B. et al. 1999, PNAS 96:2497) have demonstrated that the phenotype of complete loss of presenilin activity corresponds very well with the complete elimination of Notch signaling in the organism, suggesting that presenilins are absolutely required for Notch signaling activity. Notch receptors are single pass transmembrane proteins present at the cell surface that mediate cell—cell signaling events critical to the differentiation of many embryonic and adult tissues in invertebrates and vertebrates. Signaling involves ligand-dependent cleavage of Notch at the inner face of the transmembrane segment, and subsequent nuclear translocation of the C-terminal domain. Analysis of Notch processing in cell culture and in vivo has further demonstrated that presenilins are required for the ligand dependent cleavage event that releases the Notch intracellular domain from the transmembrane domain (Struhl, G. et al. 1999, Nature 398: 522; De Strooper, B. et al. 1999 Nature 398: 518). The parallel requirement for presenilin in both the Notch and APP cleavages suggests that the Notch signaling pathway could be a useful surrogate assay in place of Ab production in screens for presenilin pathway genes.
Mutations in the C. elegans presenilins sel-12 and hop-1 result in phenotypes associated with defective signaling by the C. elegans Notch receptors lin-12 and glp-1. Loss of hop-1 alone results in no obvious phenotypes. Loss of sel-12 results in a strong egg-laying defective phenotype and vulval defects reminiscent of lin-12 mutations. Loss of both sel-12 and hop-1 produces more severe Notch phenotypes that seen in sel-12 alone. The specific phenotypes observed in the sel-12; hop-1 double mutants depends on whether these worms inherit maternal wild type presenilin activity. When maternally provided sel-12+ activity is present, the double mutant displays a novel egg-laying defective phenotype and all progeny arrest during embryogenesis with glp-1-like developmental defects. In the absence of maternal sel-12+ activity the double mutant exhibits a stonger phenotype of sterility with germline proliferation defects characteristic of glp-1 mutants. Together, this set of properties indicates that sel-12 and hop-1 are partially redundant and act coordinately to promote signaling by the two C. elegans Notch receptors.
The partial redundancy between sel-12 and hop-1 activities made it possible to look for enhancers of sel-12 loss of function alleles that would produce a phenotype equivalent to the sel-12; hop-1 double mutant. This enhancer screen identified two new genes which were named pen-1 and 2 (pen=presenilin enhancer) and which are required for presenilin function. Based on the phenotypes of the pen genes, we have identified a third presenilin enhancer gene, aph-2. The pen-1, pen-2 and aph-2 gene sequences identify orthologous genes in humans and other animals, including pen-1B. These genes and the processes they regulate are targets for the development of therapeutics for the treatment of Alzheimer's disease.